A Thrombin specific antibody has been precoated onto 96-well plates and blocked. Standards or test samples are added to the wells and subsequently a Thrombin specific biotinylated detection antibody is added and then followed by washing with wash buffer. Streptavidin-Peroxidase Conjugate is added and unbound conjugates are washed away with wash buffer. TMB is then used to visualize Streptavidin-Peroxidase enzymatic reaction. TMB is catalyzed by Streptavidin-Peroxidase to produce a blue color product that changes into yellow after adding acidic stop solution. The density of yellow coloration is directly proportional to the amount of Thrombin captured in plate.

The thrombin ELISA, using the Abcam human ELISA kit Ab108909, lot GR232366-8, was performed by an experienced laboratory technician at an accredited university. The goal of the experiment was to analyze the linearity of dilutions of rhesus macaque EDTA-prepared plasma samples and serum samples.

Overall, the kit, like other Abcam ELISA kits, was easy to follow, clear in terms of directions, and brief yet thorough. The laboratory technician had no problems understanding any of the protocol’s steps.

Our test was performed in 2 basic steps. The first was to find the appropriate testable dilution in plasma and serum, and the second was to test the linearity and spike recovery.

To this end our first experiment tested 8 dilutions of both serum and plasma ranging from 1:10 to 1:163840 (8 1:4 serial dilutions). The plasma was not in range of the assay, confirming that his kit does not detect pro-thrombin. The serum dilutions gave OD values in range of the assay from 1:10 – 1:2560, but a linear range was not observed.

We saved the thrombin standard vial at -80C for 1 week between tests.

Our second experiment consisted of a narrower dilution range at 1:50, 1:100, 1:200, 1:400,a nd 1:800 for n=2 rhesus sera and a spike recovery test. The tested range was well within the OD limits of the assay. N=1 rhesus serum may have been near linear range at 1:400-1:800, but n=1 rhesus serum was not. The spike recovery (performed with Serum from animal 2) showed an increasing spike recovery with dilution (40% at 1:50, 80% at 1:800).

Overall, we would recommend this kit for its ease of use. We believe there is positive cross-reactivity for rhesus, but we were unable to identify a linear range for the assay.

Thank you for calling Abcam earlier today.I have talked to the lab and they do not think there is protein the same size as thrombin in the buffer based, but we cannot be 100% sure. The exact components of the buffer are proprietary and so I am una...